Cellulosic and lignocellulosic materials are major constituents of plant biomass, and cellulose polymers found therein can provide a significant source of glucose or other fermentable mono- and oligosaccharides that can in turn be metabolised by solventogenic microorganisms to produce useful solvents, such as ethanol, acetone or butanol.
Cellulose polymers can be hydrolysed by cellulose-depolymerising enzymes commonly known as cellulolytic enzymes or cellulases. For example, hydrolysis of native cellulose mainly involves four cellulase types: cellobiohydrolase (1,4-β-D-glucan cellobiohydrolase, EC 3.2.1.91), endo-β-1,4-glucanase (endo-1,4-β-D-glucan 4-glucanohydrolase, EC 3.2.1.4), endo-processive cellulases (EC 3.2.1.4./3.2.1.91), and β-glucosidase (EC 3.2.1.21). Cellulases and related enzymes have been widely utilised in various areas of biotechnology including in food, beer, wine, animal feeds, textile production and laundering, pulp and paper industry, agricultural industry and others (for review see Bhat 2000. Biotechnical Advances 18: 355-383).
Cellulases can vary in their characteristics, such as inter alia they may act as endoglucanases or as processive exoglucanases, they may generate monomers or oligomers of various lengths, they may have contrasting abilities to hydrolyse distinct cellulose forms such as crystalline cellulose, semi-crystalline cellulose, amorphous cellulose or hemicellulose, they may further display different strength of binding to cellulose substrates, different kinetic parameters, etc. Therefore, significant effort needs to be invested into further characterisation of cellulases, so as to identify functional parts or domains thereof which may underlie the interesting properties of these enzymes. Such functional domains can be advantageously combined with other cellulases or domains thereof to generate chimeric enzymes with desired activities.
Further, the recombinant expression of cellulases in solventogenic microorganisms—to allow the production of useful solvents including inter alia ethanol by these microorganisms directly from cellulose-containing materials—is not yet satisfactorily advanced. To achieve further improvements, cellulases particularly suitable for or advantageous in such applications need to be recognised, characterised and selected.
Saccharophagus degradans strain 2-40, previously known as Microbifulber degradans strain 2-40 and deposited with the American Type Culture Collection under accession number ATCC 43961, is a marine γ-proteobacterium able to degrade at least ten different complex polysaccharides including inter alia cellulose (Andrykovitch & Marx 1988. Appl Environ Microbiol 54: 3-4; Ensor et al. 1999. J Ind Microbiol Biotechnol 23: 123-126).
The S. degradans genome has been sequenced and its cellulase system identified, including among many others a cellulase denoted as Cel5H (Taylor et al. 2006. J Bacteriol 188: 3849-61). Further, US2007/0292929 generically contemplates recombinant micro-organisms including ethanologenic bacteria expressing the cellulose degrading proteins from S. degradans. However, only the activity of enzymes expressed by S. degradans is demonstrated and it is unclear whether any of these enzymes would function appropriately when expressed in ethanologenic bacteria.